Plasticized bone grafts, and methods of making and using same

ABSTRACT

The present invention provides a plasticized dehydrated or freeze-dried bone and/or soft tissue product that does not require special conditions of storage, for example refrigeration or freezing, exhibits materials properties that approximate those properties present in normal hydrated tissue, is not brittle, does not necessitate rehydration prior to clinical implantation and is not a potential source for disease transmission. The invention replaces water in the molecular structure of the bone or soft tissue matrix with one or more plasticizers allowing for dehydration of the tissue, yet not resulting in an increase in brittleness of the plasticized product, and resulting in compressive and/or tensile properties similar to those of normal hydrated bone. Replacement of the chemical plasticizers by water prior to implantation is not required and thus, the dehydrated bone or soft tissue plasticized product can be placed directly into an implant site without significant preparation in the operating room.

This application is a continuation application of U.S. patentapplication Ser. No. 09/107,459 filed Jun. 30, 1998, now allowed.

FIELD OF THE INVENTION

The present invention provides a plasticized dehydrated bone and/or softtissue product that does not require special conditions of storage, forexample refrigeration or freezing, exhibits materials properties thatapproximate those properties present in normal hydrated tissue, is notbrittle and does not necessitate rehydration prior to clinicalimplantation. The invention replaces water in the molecular structure ofthe bone or soft tissue matrix with one or more plasticizers allowingfor dehydration of the tissue, yet not resulting in an increase inbrittleness of the plasticized product, and resulting in compressiveand/or tensile properties similar to those of normal hydrated bone.Replacement of the chemical plasticizers by water prior to implantationis not required and thus, the dehydrated bone or soft tissue plasticizedproduct can be placed directly into an implant site without significantpreparation in the operating room. The present plasticized graft doesnot need rehydration, possesses adequate materials properties, and isnot a potential source for disease transmission.

BACKGROUND OF THE INVENTION

Bone tissue is a homogeneous material comprised of osteoid and minerals.The osteoid is a viscous gel-like material comprised primarily of type Icollagen (approximately 90%), proteoglycans, and various sulfated andnon-sulfated mucopolysaccharides. The mineral component consistsprimarily of a crystalline form of calcium phosphate, hydroxy apatite,with amounts of calcium carbonate, tricalcium phosphate, and smalleramounts of other forms of mineral salts. This bone tissue is laid downaround cells called osteocytes and these cells are found in smallinterconnected channels (lacunnae) which are interconnected through aseries of channels comprising the Haversian canal system. At the levelof the microscope, it is possible to observe that bone tissue isorganized into osteons of compact bone made of concentric, perivascularlayers of highly coaligned mineralized collagen fiber bundles. Thepredominant orientation within a single layer varies with respect to thevascular axis and various combinations of orientation in successivelamellae result in variable overall collagen orientation within eachosteon. Differences in overall collagen orientation are directlyreflected in differing mechanical behavior of single osteons.Transversely oriented collagen results in better resistance tocompressive loading along the axis, whereas predominant longitudinalorientation results in better resistance to tensile stress. Thepredominant orientation of collagen within a cross-section of long boneis not random, but matches the expected distribution of mechanicalstress across the section, and its rotational shift along the wholeshaft. More transverse collagen is deposited at sites of compressiveloading, and more longitudinal collagen is deposited at sites of tensilestress. These structural oriented bone tissues in a load bearing boneare presumed to be laid down by the osteocytes present in the bone andbone remodeling mediates mechanical adaptation in compact bone.

A bone is typically comprised of bone tissue in the form of cortical andtrabecular bone. Cortical bone is frequently referred to as compact boneand is the major load-bearing part of a bone. Trabecular bone is presentin what is typically referred to as cancellous bone where it appears asa densely interconnected structure of “spongy” bone. Spongy bone in atypical bone contains the hemotopoietic cellular elements which iscalled bone marrow. Trabecular bone can be described as forming across-bracing lattice between cortical bone in a bone. It is importantto emphasize a need to differentiate between “a bone” and “bone” (as atissue). A bone is comprised of bone tissue present as cortical andcancellous (spongy) bone.

The mineralized osteoid typical of bone tissue is hydrated along theorganic molecular structure and is an essential element of the mineralstructure. Hydrating molecules of water form complex molecularassociations with these organic and non-organic elements of bone tissueand can be described as being tightly bound, loosely bound, and free.Free water and loosely bound water can frequently be removed from bonetissue with only minor changes in the overall mechanical characteristicsof the bone tissue. Tightly bound water can be removed only underextreme conditions and results in significant changes in the physicaland mechanical properties of bone tissue. In fresh bone, water serves asolvating function in bone tissue allowing proper orientation andmolecular spacing of the collagen fibrils which maintain structuralalignment of the mineral phase in association with the organic phase.

Bone tissue in the form of bone grafts for implantation into a patient,is typically preserved and provided in a dehydrated state. Dehydrationof bone tissue through drying, whether by air drying or sublimation asin freeze-drying, results in alteration of the molecular structure ofthe bone tissue and as a result of the reorientation of the collagenfibrils and the crystalline mineral phase, stress accumulates in thebone tissue. This stress can be relieved by rehydration or by theoccurrence of small or large dislocations of structure. Smalldislocations are designated micro fractures and are not usually visibleto the naked eye. Large dislocations are designated fractures and areusually visible to the naked eye.

In a long bone, for example a femur, tibia, fibula, or humerus, theshaft separates the proximal and distal ends of the long bone. The shaftserves to focus loads applied to the whole bone into a smaller diameterthan found at the proximal and distal ends of the long bone and theshaft of a long bone is typically of a cylindrical shape and iscomprised of compact (cortical) bone. Loads applied along the axis ofthe shaft require that the cortical bone maintain a constantcircumference, i.e. the tendency to failure would distort the bonetissue perpendicular to the axis of load application. Thus, theorientation of the collagen fibers should be such that tensile stress isresisted along the axis of loading and compressive stress is resistedperpendicular to loading. Drying of shaft portions of long bones resultsin reorientation of collagen fibers and the mineral phase such thatchanges in the circumferential orientation create stress within the bonematrix which can be relieved only by rehydration or occurrence of afracture which allows a reorientation approximating the originalorientation. In dehydrated cortical ring grafts cut from shafts of longbones, this stress release can present as a fracture along the long axisof the bone shaft leaving a circumference which approximates thecircumference of the cortical ring graft prior to drying. By rehydratingbone grafts prior to implantation, the potential for fracture formationwhich can compromise the function of the bone product can be reduced,but not eliminated. Fractures as discussed above can occur in dehydratedbone prior to rehydration and result in a graft having compromisedbiomechanical properties, which in turn can result in graft failure whenimplanted in a patient.

Load-bearing soft tissue grafts such as ligaments, tendons, and fascialata are frequently provided in a freeze-dried state. Such grafts mustbe rehydrated prior to clinical implantation. Such soft tissue graftstypically contain collagen, elastin, and assorted proteoglycans andmucopolysaccharides. The collagens and elastins are the load-bearingcomponent(s) of these soft tissue grafts and the assorted proteoglycansand polysaccharides serve to bind the fibrillar collagens into amatrix-like structure. The structural organization of fascia lata issimilar to dura mater in being somewhat isotropic in load-bearingproperties (Wolfinbarger, L, Zhang, Y, Adam, B L T, Homsi, D, Gates, K,and Sutherland, V, 1994, “Biomechanical aspects on rehydratedfreeze-dried human allograft dura mater tissues, J. AppliedBiomaterials, 5:265-270) whereas tendons (for example the Achillestendon) or ligaments (for example the Anterior cruciate ligament) aretypically anisotropic in load-bearing properties. In these types ofload-bearing soft tissue grafts, the tensile properties of the tissuesdepend on the flexibility of the collagenous structures to stretch underload and return to their original dimensions upon removal of the load.

A wide variety of bone and soft tissue products are used in veterinary,medical, orthopaedic, dental, and cosmetic surgery applications. Thesebone and soft tissue products can be used in load-bearing and non-loadbearing applications and the bone and soft tissue products can besupplied under a variety of forms. Bone products are provided asfresh-frozen, freeze-dried, rehydrated freeze-dried, air-dried, organicsolvent preserved, or provided preserved by other similar types ofpreservation methods. Each method of preservation of bone productspossesses selected advantages and disadvantages and thus the method ofpreservation is generally modified to select for specific needs of agiven bone graft. Soft tissue products are typically provided asfresh-frozen or freeze-dried and each method of preservation of softtissue products possess selected advantages and disadvantages and thusthe method of preservation is generally modified to select for specificneeds of a given soft tissue product.

Bone and soft tissue products preserved and stored by methods involvingfreeze-drying (removal of water by sublimation) yield a bone or softtissue product which is significantly more brittle than normal bone andhas a tendency to fracture into numerous small pieces, which ultimatelycan result in graft failure. Specifically, freeze-drying causes graftsto be brittle and typically causes shrinkage where the shrinkage isoften not uniform, thereby causing graft failure; solvent preservationusing for example, acetone or alcohol, can cause irreversibledenaturation of proteins, and solubilization of solvent solublecomponents, including for example, lipids. These alterations inmaterials properties of the bone and soft tissue products necessitates arehydration step in preparation of the bone and soft tissue product forimplantation. However, rehydration does not solve the problem, graftscan fracture prior to rehydration, thereby making rehydration futile,and if there are micro fractures prior to rehydration they remain afterrehydration. These grafts are more likely to fail regardless of whetherthey are rehydrated. Even after rehydration the materials properties donot approximate the materials properties of normal bone.

Bone and soft tissue products are generally separated into load bearingand non-load bearing products. Examples of non-load bearing boneproducts are ground demineralized bone which are used for inducing newbone formation in a particular implant site. Load-bearing bone productsare rarely demineralized and are used at implant sites where the bonegraft will be expected to withstand some level of physical load(s). Itis therefore important that load bearing bone products not fail duringnormal movement(s) of the implant recipient and that the bone productnot stimulate a pronounced physiological response. The majority of boneproducts are provided in either the fresh-frozen or freeze-dried format.The fresh-frozen format is undesirable because it includes donor derivedbone marrow and is thus immunogenic and a source of diseasetransmission. The freeze-dried format is less of a problem thanfresh-frozen grafts in the potential for disease transmission, however afreeze-dried bone graft is significantly more brittle than normal bone,more brittle than fresh frozen bone, and must be rehydrated prior toclinical usage. In that clinicians typically do not have time toadequately rehydrate bone graft products in the operating room, it isadvantageous to provide a dehydrated or freeze-dried bone product whichdoes not need rehydration, possesses adequate materials properties, andis not a potential source for disease transmission.

SUMMARY OF THE INVENTION

It is an objective of the present invention to provide implantable,non-demineralized, load-bearing bone products which are mechanicallystabilized in a dehydrated state by use of biocompatible plasticizers.

It is a further objective of the present invention to provideimplantable, load-bearing, soft tissue products which are mechanicallystabilized in a dehydrated state by use of biocompatible plasticizers.

It is also an objective of the present invention to provide implantable,load-bearing, bone products which do not require rehydration.

It is yet a further objective of the present invention to provideimplantable, load-bearing, soft tissue products which do not requirerehydration.

It is an objective of the present invention to provide methods ofplasticizing load-bearing bone and soft tissue products.

It is a further objective of the present invention to provideplasticized bone and soft tissue products which are resistant toproliferation of microorganisms.

It is yet a further objective of the present invention to provide boneand soft tissue products which can be stored at room temperature usingconventional packaging.

It is a further objective of the present invention to provideplasticized bone and soft tissue products where the plasticizer can bereadily removed prior to implantation.

It is a further objective of the present invention to use plasticizersto plasticize bone and soft tissue products which are not toxic to arecipient of the plasticized bone or soft tissue graft.

It is yet a further objective of the present invention to provideimplantable load-bearing bone and soft tissue products which are similarin physical, chemical, and biological properties as compared to normaltissue (fresh bone or fresh soft tissues) yet lack the inherentdisadvantages (including for example, potential disease transmission,increased immunogenicity, and a tissue (e.g. bone marrow) which canyield toxic degradation products and/or retard graft incorporation) offresh-frozen, dehydrated, and freeze-dried bone and/or soft tissueproducts.

It is a further objective of the present invention to provide aplasticized bone graft suitable for transplantation into a human,including a non-demineralized bone graft having an internal matrixessentially free from bone marrow elements; and one or more plasticizerscontained in the internal matrix.

It is an object of the present invention to provide a plasticized bonegraft, including a cleaned, non-demineralized, bone graft; and one ormore plasticizers, where the cleaned non-demineralized bone graft isimpregnated with the one or more plasticizers.

It is yet a further objective of the present invention to provide aplasticized bone graft, including a cleaned, non-demineralized, bonegraft including one or more plasticizers.

It is a further objective of the present invention to provide a methodfor producing a plasticized bone graft suitable for transplantation intoa human, by impregnating a cleaned, non-demineralized, bone graft withone or more plasticizers to produce a plasticized bone graft.

Plasticity of soft tissues depends primarily on the waters of hydrationpresent in the matrix structure, where water movement under a load isrestricted by the viscous nature of the proteoglycan/polysaccharidecomponent, and bound waters of hydration in the collagen componentaffect the flexibility of the tensile component of the tissue(s). Thepresent invention deals with the plasticization of these load bearingtissue constructs where the water removed is replaced with one or moreplasticizers including for example, glycerol (glycerin USP) (liquidsubstitution) such that the graft does not need to be rehydrated orwashed to remove the plasticizer prior to clinical implantation.

The present invention provides a dehydrated or freeze-dried plasticizedbone or soft tissue product, preferably containing less than 5% residualmoisture, which product requires no or minimal processing just prior toclinical implantation. The present invention solves prior art problemsof grafts having insufficient materials properties, graft brittleness,and the necessity for rehydration prior to clinical implantation, byproviding a plasticized dehydrated bone and/or soft tissue product thatexhibits materials properties that approximate those properties presentin normal hydrated tissue, is not brittle and does not necessitaterehydration prior to implantation.

DETAILED DESCRIPTION

I. Definitions

The below definitions serve to provide a clear and consistentunderstanding of the specification and claims, including the scope to begiven such terms.

Alcohol. By the term “alcohol” is intended for the purposes of thepresent invention, one of a series of organic chemical compounds inwhich a hydrogen attached to carbon is replaced by a hydroxyl. Suitablealcohols useful in the plasticizer composition of the present inventionpreferably include C₁-C₁₀ alcohols, and more preferably ethanol andisopropyl alcohol.

Allowash™ Solution. By the term “Allowash™ Solution” is intended thosedetergent compositions disclosed in co-pending U.S. patent applicationSer. No. 08/620,856 incorporated herein by reference. Examples ofsuitable Allowash compositions include: a cleaning compositioncontaining essentially about 0.06 wt % polyoxyethylene-4-lauryl ether,about 0.02 wt % poly (ethylene glycol)-p-nonyl-phenyl-ether; about 0.02wt % octyphenol-ethyleneoxide and endotoxin free deionized/distilledwater.

Biocompatible. By the term “biocompatible” is intended for the purposesof the present invention, any material which does not provoke an adverseresponse in a patient. For example, a suitable biocompatible materialwhen introduced into a patient does not itself provoke a significantimmune response, and is not toxic to the patient.

Biomechanical strength. By the term “biomechanical strength” is intendedfor the purposes of the present invention, those properties exhibited bya tissue graft, including loading strength, compressive strength, andtensile strength.

Bone graft. By the term “bone graft” is intended for the purposes of thepresent invention, any bone or piece thereof obtained from a donor forexample a human or animal and/or cadaver donor, including for exampleany essentially intact bone graft including for example the femur,tibia, ilia, humorous, radius, ulna, ribs, whole vertebrae, mandibulaand/or any bone which can be retrieved from a donor with minimal cuttingof that bone for example, one half of an ulna, a femur cut in half toyield a proximal half and a distal half, femoral head, acetabula, distalfemur, femur shaft, hemi-pelvi, humerus shaft, proximal femur, proximalfemur with head, proximal humeri, proximal tibia, proximaltibia/plateaus, talus, tibia shaft, humeral head, ribs, and/or at leasta substantial portion of a whole bone, i.e. at least one-quarter of awhole bone; and/or any cut bone grafts including for example an iliaccrest wedge, a Cloward dowel, a cancellous cube, a fibular strut,cancellous block, a crock dowel, femoral condyles, femoral ring, femursegment, fibula segment, fibular wedge, tibia wafer, ilium strip, MidasRex dowel, tibial segment, and radius/ulna wedge.

Bone marrow elements. By the term “bone marrow elements” is intended forthe purposes of the present invention, the highly cellular hematopoieticconnective tissue filling the medullary cavities and spongy epiphysis ofbones which may harbor bacterial and/or viral particles and/or fungalparticles, and includes for example, blood and lipid.

Cleaned bone graft. By the term “cleaned bone graft” is intended for thepurposes of the present invention, a bone graft that has been processedusing means know in the art, to remove bone marrow elements.

Dehydrated bone or soft tissue. By the term “dehydrated bone or softtissue” is intended bone tissue or soft tissue which is preserved bydehydration, such drying methods including for example, freeze-drying,and/or sublimation and/or air drying and/or liquid substitution.

Essentially free from. By the term “essentially free from” is intendedfor the purposes of the present invention, a bone graft where thematerial removed (i.e., bone marrow elements) from the bone graft is notdetectable using detection means known in the art at the time of filingof this application.

Incubating. By the term “incubating” is intended for the purposes of thepresent invention, processing a bone graft in for example a plasticizercomposition by soaking the graft in the composition, shaking the graftwith the composition, subjecting the graft to flow of the compositionwhere the flow is induced by negative or positive pressure, subjectingthe graft and/or the composition to negative or positive pressure, orsoaking the bone graft in a plasticizer composition in a negativepressure environment.

Impregnating. By the term “impregnating” is intended for the purposes ofthe present invention, any processing conditions which result in fillingthe internal matrix of a bone graft with a plasticizer composition.

Internal matrix. By the term “internal matrix” is intended for thepurposes of the present invention, the spongy epiphysis of bones, theintercellular substance of bone tissue including collagen fibers andinorganic bone salts; or in soft tissue, the intercellular substance ofsuch soft tissue including for example ligaments and tendons, includingcollagen and elastin fibers and base matrix substances.

Load-bearing. By the term “load-bearing” is intended for the purposes ofthe present invention a non-demineralized bone product or soft tissueproduct for implantation in a patient at a site where the bone graft orsoft tissue graft will be expected to withstand some level of physicalload(s).

Materials properties. By the term “materials properties” is intended forthe purposes of the present invention, those properties present innormal fresh bone which include for example, loading strength,compressive strength, tensile strength, and deformability.

Negative pressure. By the term “negative pressure” is intended for thepurposes of the present invention, a pressure below atmosphericpressure, i.e. below 1 atm.

Normal bone or soft tissue. By the term “normal bone or soft tissue” isintended for the purposes of the present invention, fresh hydratedautogenous and/or fresh-frozen hydrated allograft tissue including forexample, bone, fascia, ligaments, and tendons.

Permeation enhancer. By the term “permeation enhancer” is intended forthe purposes of the present invention, any agent including for example,isopropyl alcohol, that facilitates penetration of the one or moreplasticizers or plasticizer composition into the bone or soft tissue. Inthe case of isopropyl alcohol, permeation is enhanced due to the reducedsurface tension of the alcoholic solution.

Plasticization. By the term “plasticization” is intended for thepurposes of the present invention, replacing free and loosely boundwaters of hydration in a tissue(s) with one or more plasticizers withoutaltering the orientation of the collagen fibers and associated mineralphase.

Plasticizer. By the term “plasticizer” is intended for the purposes ofthe present invention, any biocompatible compounds which are soluble inwater and can easily displace/replace water at the molecular level andpreferably have a low molecular weight such that the plasticizer fitsinto the spaces available to water within the hydrated molecularstructure of the bone or soft tissue. Such plasticizers are preferablynot toxic to the cellular elements of tissue into which the graft is tobe placed, or alternatively, the plasticizer is easily removed from thegraft product prior to implantation. Suitable plasticizers arepreferably compatible with and preferably readily associates with themolecular elements of the bone tissue and/or soft tissue. Suitableplasticizers include for example: glycerol (glycerin USP), adonitol,sorbitol, ribitol, galactitol, D-galactose, 1,3-dihydroxypropanol,ethylene glycol, triethylene glycol, propylene glycol, glucose, sucrose,mannitol, xylitol, meso-erythritol, adipic acid, proline, hydroxyprolineor similar water-soluble small molecular weight solutes which can beexpected to replace water in the base matrix structure of bone tissueand/or soft tissue and provide the hydrating functions of water in thattissue. Suitable solvents include for example: water, alcohols,including for example ethanol and isopropyl alcohol.

Plasticizer composition. By the term “plasticizer composition” isintended for the purposes of the present invention, any compositionwhich includes one or more plasticizers and one or more biocompatiblesolvents. Suitable solvents include for example: water, and alcohols,including for example C₁-C₁₀ alcohols, and more preferably ethanol andisopropyl alcohol.

Positive pressure. By the term “positive pressure” is intended for thepurposes of the present invention, a pressure above atmosphericpressure, i.e. above 1 atm.

Rehydration. By the term “rehydration” is intended for the purposes ofthe present invention, hydrating a dehydrated plasticized tissue graftor a dehydrated non-plasticized tissue graft, with water, for example,prior to implantation into a patient. In the case of a plasticizedgraft, the plasticizer may optionally be not replaced by water or mayoptionally be partially or fully replaced by water.

Soft tissue grafts. By the term “soft tissue grafts” is intended for thepurposes of the present invention, load-bearing and non-load-bearingsoft tissue products. Non load-bearing grafts include cadaveric skin.Load-bearing soft tissue grafts include for example: pericardium, duramater, fascia lata, and a variety of ligaments and tendons. Soft tissuegrafts are composed of an internal matrix which includes collagen,elastin and high molecular weight solutes where during cleaning cellularelements and small molecular weight solutes are removed.

II. Plasticizers

Plasticization of load-bearing bone or soft tissue grafts represents amethod of replacing free and loosely bound waters of hydration in thetissue(s) with a plasticizer composition containing one or moreplasticizers, without altering the orientation of the collagen fibersand associated mineral phase. Suitable plasticizers include compoundswhich are soluble in water and can easily displace/replace water at themolecular level. Suitable plasticizers preferably have a low molecularweight such that the plasticizer fits into the spaces available to waterwithin the hydrated molecular structure of the bone or soft tissue. Suchplasticizers are not toxic to the cellular elements of tissue into whichthe graft is to be placed, or alternatively, the plasticizer is easilyremoved from the graft product prior to implantation. Finally, theplasticizer is preferably compatible with and preferably readilyassociates with the molecular elements of the bone or soft tissue.

Plasticizers suitable for use in the present invention include forexample, a variety of biocompatible aqueous solutions. Examples ofacceptable plasticizers include, but are not restricted to, members ofthe polyol family (sugar alcohols) of compounds including C₂ to C₇polyols, monoglycerides (such as monoolein and monolinolein), andvarious short- and medium-chain free fatty acids (such short-chain freefatty acids preferably having a carbon chain length of less than sixC₆), and such medium-chain free fatty acids preferably having a carbonchain length of from C₁₂ to C₁₄) and their correspondingmonoacylglycerol esters (MGs) such as the saturated MGs, ranging incarbon chain length from C₅ to C₆, and preferably C₅ to C₁₄ MGs.Specific plasticizers include, but are not limited to, glycerol(glycerin USP), adonitol, sorbitol, ribitol, galactitol, D-galactose,1,3-dihydroxypropanol, ethylene glycol, triethylene glycol, propyleneglycol, glucose, sucrose, mannitol, xylitol, meso-erythritol, adipicacid, proline, hydroxyproline or similar water-soluble small molecularweight solutes which can be expected to replace water in the base matrixstructure of bone or soft tissue, and provide the hydrating functions ofwater in that tissue. Other plasticizers suitable for use in the presentinvention can be readily selected and employed by one of ordinary skillin the art to which the present invention pertains without undueexperimentation depending on the desired clinical outcome, sensitivityof the implantation procedure, patient sensitivities, and physicianchoice.

The present plasticizers are preferably employed at a concentration inthe range of from 0.1 to 2.0 M, 10% to 100% by weight/volume, or 3% to30% by weight of bone or soft tissue. The use of Molar concentrationsand weight/volume percentages to express preferred concentration rangesare intended to deal with the concentrations of these plasticizers inthe solutions used to treat the tissues. The use of the weight percentof plasticizer in load-bearing bone or soft tissue is intended to dealwith the effective quantity of a given plasticizer in the load-bearingtissue which is necessary to effectively replace the waters of hydrationpresent in the unprocessed tissues which are maximally plasticized to astate approximating normal tissue. The plasticizer can be introducedinto the bone or soft tissue matrix at any number of steps in theprocessing procedures and at a variety of concentrations with andwithout the use of permeation enhancers.

The result(s) of plasticization of load-bearing bone and soft tissueproducts are bone or soft tissue products which are similar totraditionally dehydrated bone and soft tissue products in residualmoisture but are not subject to fractures or micro fractures like suchdehydrated products, yet do not need to be rehydrated prior to use. Themechanical and use properties of a plasticized bone or soft tissueproduct are similar to those of natural (fresh autogenous and/orfresh-frozen allograft) bone, dura, pericardium, fascia, ligaments, andtendons.

III. Graft Cleaning and Processing

The present plasticizers may be introduced to the bone or soft tissueproducts at several points in the processing procedure(s). Boneprocessing and cleaning procedures suitable for use with the presentinvention include known processes, as well as the processes described inU.S. Pat. No. 5,556,379 and co-pending U.S. patent application Ser. Nos.08/871,601 “Process for Cleaning Grafts Using Centrifugal Force and BoneGrafts Produced Thereby”; Ser. No. 08/620,858 “Composition for CleaningBones”; Ser. No. 08/646,520 “Recirculation Method for CleaningEssentially Intact Bone Grafts Using Pressure Mediated Flow of Solutionsand Bone Grafts Produced Thereby”; and Ser. No. 08/646,519 “UltrasonicCleaning of Allograft Bone” which are hereby incorporated herein intheir entirety. The plasticizers may be incorporated into the processingprocedure(s) using steps where the plasticizer(s) is/are present atessentially full strength, i.e. 100% concentration, in the presenceand/or absence of permeation enhancers, and at concentrations less thanfull strength.

Bone tissue is cleaned and processed as described in U.S. Pat. No.5,556,379, and co-pending U.S. patent application Ser. Nos. 08/871,601;08/620,858; 08/646,520; and 08/646,519 by for example, transection of anessentially intact bone or perforation of an essentially intact bonewith attachment of sterile plastic tubing to the cut end of a transectedbone or to an attachment port inserted into the perforation of theperforated bone. The bone is immersed in a cleaning solution, suchsolutions including known cleaning agents as well as those described inthe above-identified patent and co-pending patent applications, with orwithout use of sonication. The cleaning solution is induced to flowinto, through, and out of the bone through use of a peristaltic pump ornegative pressure applied to the cleaning solution, The induced flow ofcleaning solution draws the bone marrow from the interior of the bone,and particularly from the cancellous bone marrow space, where it can besafely deposited in a receiving container containing a strong virucidalagent such as sodium hypochlorite (common bleach). The cleaned bone canthen be further cleaned by causing the cleaning solution to be replacedwith a solution of one or more decontaminating agents, including forexample 3% hydrogen peroxide, with or without plasticizer. Hydrogenperoxide which in addition to its mild disinfection activity generatesoxygen bubbles that can further assist in dislodging residual bonemarrow materials causing the residual bone marrow materials to flow fromthe bone and into the receiving container.

In the above-described process, after processing with the cleaningsolution, after processing with a decontaminating agent, in place ofprocessing with a decontaminating agent, or after dehydration, thecleaned graft is plasticized for example, by processing the cleanedgraft with a plasticizer composition containing one or more plasticizersincluding for example glycerin USP in a solvent.

IV. Plasticization

Bone and soft tissue grafts can be cleaned and processed usingconventional methods including those described in. When processing usingthese methods the graft is plasticized by adding one or moreplasticizers or a plasticizer composition to processing steps after bonecleaning is essentially completed, and prior to freeze-drying. Underfreeze-drying, the water present in the bone (or smaller cut bone graftsproduced form the essentially intact bone) is removed by sublimation,however, the glycerol will remain and replace the free and bound wateras the water is removed from the bone tissue. The one or moreplasticizer(s) is added to fully hydrated bone tissue and theplasticizer(s) are induced to penetrate into the bone tissue optionallyusing a permeation enhancer. Thus, the bone or soft tissue is dehydratedyet the materials properties of the bone tissue will be similar to thematerials properties of normal bone or soft tissue, i.e. partially orfully hydrated bone or soft tissue. The produced plasticized bone orsoft graft contains minimal quantities of the plasticizer(s) and can beremoved from the package and directly implanted into a patient withoutrehydration. If the presence of these small quantities of glycerol is ofconcern, the bone or soft tissue grafts may be quickly rinsed and/orwashed in sterile saline just prior to implantation.

Bone or soft tissue cleaned and processed by the methods as describedfor bone cleaning and processing in U.S. Pat. No. 5,556,379, and/orco-pending U.S. patent application Ser. Nos. 08/871,601; 08/620,858;08/646,520; and/or 08/646,519 and/or bone or soft tissue cleaned andprocessed by conventional methods, may be plasticized by processing withthe plasticizer composition containing one or more plasticizers,including for example glycerin USP, in a solvent by for example drawingthe plasticizer composition into the bone. Suitable solvents include forexample, 70% isopropyl alcohol. The 70% isopropyl alcohol/plasticizercomposition can be prepared by diluting absolute (100%) isopropylalcohol with the one or more plasticizers, including for exampleglycerin USP such that the plasticizer accounts for 30% of the totalvolume and isopropyl alcohol accounts for 70% of the total volume. Underthis method, the original processing procedures as described in U.S.Pat. No. 5,556,379 regarding the use of 70% isopropyl alcohol, isretained essentially unchanged. The isopropyl alcohol facilitatespenetration of the glycerol into the tissue by acting as a permeationenhancer and the glycerol more readily penetrates the tissue due to thereduced surface tension of the alcoholic solution. The induced flow ofglycerol/isopropyl alcohol into, through, and out of for example, theessentially intact bone, further serves to remove residual cellularelements, for example bone marrow materials, if any. It also allowspenetration of the glycerol/isopropyl alcohol solution into the mostremote areas of the tissue, and facilitates a uniform distribution ofthe glycerol into the tissue. The isopropyl alcohol can be removed fromthe tissue by washing with a washing solution including sterile water,for example as described in U.S. Pat. No. 5,556,379 following thealcohol processing step. Preferably, the washing solution includesglycerin USP (30% volume:volume). The washing solution facilitatesremoval of the isopropyl alcohol without removal of the glycerin USP.The cleaned and plasticized tissue can then be frozen and freeze-driedor dehydrated according to standard protocols.

Alternatively, bone or soft tissue grafts may be plasticized aftercleaning and freeze-drying. For example, tissue can be processed andcleaned according to any method including known methods, or as describedin U.S. Pat. No. 5,556,379 described above. After the sterile water washthe tissue (for example bone tissue) is cleaned of virtually allcellular elements (for example, bone marrow) present in the tissue andthe cleaned tissue can be further processed into for example, small cutbone grafts, and dehydrated or freeze-dried (also called lyophilized)using standard methods well known to those skilled in the art.Freeze-dried or dehydrated tissue grafts preferably contain less thanabout 5% residual moisture, satisfying the definition of freeze-driedbone allografts as prescribed under Standards of the AmericanAssociation of Tissue Banks.

Clean freeze-dried or dehydrated bone or soft tissue grafts areplasticized by processing the tissue graft with a plasticizercomposition, suitable compositions including for example 70% isopropylalcohol/30% glycerin USP or 100% glycerin USP. Due to the presence ofair in the cancellous and cortical bone spaces, the plasticizer(s) mayonly penetrate into the bone tissue with which it is in physicalcontact. Suitable methods for achieving physical contact between theplasticizer and bone or soft tissue include those methods known to oneof ordinary skill in the art to which the present invention pertains.The plasticizer composition can be induced to flow into the cancellousand cortical bone spaces of bone tissue, or soft tissue, thus achievingphysical contact, by various known methods that can be readily selectedand employed by one of ordinary skill in the art to which the presentinvention pertains without undue experimentation, and include forexample, agitation of the a tissue with the plasticizer composition,application of a vacuum (5 to 500 mTorr) above the plasticizer. Thevacuum induces the air trapped in the, for example cancellous andcortical bone spaces/tissue to exit and be carried off. As the trappedair is removed from the cancellous and cortical bone spaces/tissue, theplasticizer quickly moves into the spaces previously occupied by airgreatly enhancing penetration of the plasticizer into the bone or softtissue. The plasticizer fills the spaces previously occupied by the freeand bound water restoring the tissue to a materials property similar tothat materials property of the original fully or partially hydratedtissue (e.g. normal bone).

The present one or more plasticizers may be introduced to soft tissueproducts at several points in the processing procedures, but arepreferably introduced prior to the freeze-drying or dehydrating step. Byintroducing plasticizers prior to freeze-drying or dehydrating, thederived soft tissue graft is in a freeze-dried/dehydrated state wherethe plasticizer is used to stabilize the matrix and load bearingcomponents of the soft tissue graft such that the graft can be usedwithout rehydration/reconstitution.

V. Transplantation into a Patient

Prior to transplantation into a patient, excess glycerol may optionallybe removed from the plasticized bone or soft tissue graft using forexample, the method described in co-pending U.S. patent application Ser.No. 08/871,601. Specifically, the plasticized grafts are placed intocentrifuge vessels/containers and on top of inserts designed to keep thebone grafts off of the bottom of the containers. The grafts are thencentrifuged at 1,000 to 2,000 revolutions per minute (rpm) for 10-20minutes. The excess glycerol or similar plasticizer exits the grafts andcollects in the bottom of the centrifuge containers away from thegrafts. The plasticizer tightly associated with the molecular andchemical structure of the tissue will not exit the graft and the tissuewill remain plasticized without retaining physically discemablequantities of plasticizer. The plasticized graft(s) may then be packageddirectly or packaged in a packaging format which permits application ofa vacuum to the container. The current value of using a packaging formatwhich permits storage of grafts under vacuum lies in the ability topredict possible loss of sterility with loss of vacuum to the packaging.

Clinical usage of plasticized bone or soft tissue grafts includes directimplantation of the grafts without further processing following removalfrom the packaging, implantation following a brief washing in sterileisotonic saline to remove any remaining traces of plasticizer associatedwith the immediate surfaces of the grafts, or by implantation followingan extended (approximately 1 hour) washing with sterile isotonic salineto remove as much plasticizer as possible. Under any of the abovedescribed further processing of grafts, the materials properties of theplasticized grafts resemble those materials properties of fully orpartially hydrated natural tissue (i.e. normal bone or soft tissue). Theproduced plasticized graft does not need to be rehydrated prior toclinical implantation, yet retains the strength and compressive/tensileproperties of natural tissue. Plasticized freeze-dried soft tissuegrafts where the plasticizer is used to stabilize the matrix and loadbearing components of the soft tissue graft, can also be directlyimplanted in a patient without rehydration/reconstitution.

Suitable surgical methods for implanting bone and soft tissue graftsinto a patient are well known to those of ordinary skill in the art towhich the present invention pertains, and such methods are equallyapplicable to implantation of the present plasticized grafts. Those ofordinary skill in the art to which the present invention pertains canreadily determine, select and employ suitable surgical methods withoutundue experimentation.

Further details of the process of the invention are presented in theexamples that follow:

EXAMPLE 1 Processing of a Frozen Distal Femur

A. Cleaning and Processing:

A frozen distal femur is selected and all of the soft tissue andperiosteum is removed using sharp dissection techniques and periostealelevators. The graft is then transected to the desired length using aStryker® saw or band saw. Each bisected piece is not more than 30 cm inlength and is straight and contains no bone fragments. The surfacecartilage is then removed from the fem oral condyle with either ascalpel blade, periosteal elevator, or osteotome. The processinginstructions dictate leaving the cartilage “on” when appropriate. Usinga ⅜″ drill bit, the cut end of the shaft is drilled approximately 5 cm.The interior of the intramedullary canal is then throughly washed withthe lavage system.

An intercalary fitting is then inserted by screwing the threaded,tapered end into the cut end of the graft. The vacuum tubing isassembled by securing one end of the tubing to the nipple end of theintercalary fitting. The other end of the tubing is secured to thepiston driven pump. Finally, another section of vacuum tubing is securedto the other side of the piston pump. Approximately 4000 cc of a 1:100dilution of the “Allowash™ Solution” is poured into the sterile flushingvessel. The “Allowash™ Solution” is prepared by adding 4 cc of cleaningreagent to 3996 cc of sterile water. The flushing vessel is labeled as“Allowash™ Solution.” The open end of the second piece of vacuum tubingis placed into a graduated flask. The piston pump is set to “reverse”and the flow rate controller is set to 50%. The pump is turned on and atleast 500 cc of the first solvent (Allowash™ Solution) is drawn towaste. Thereafter, the open end of the second piece of vacuum tubing isremoved from the graduated flask and placed into the sterile flushingvessel. The drive is maintained in the “reverse” position at 50%. TheAllowash Solution recirculates for a minimum of 15 minutes.

The 1:100 dilution of the Allowash™ Solution is then decanted andapproximately 4 liters of 3% hydrogen peroxide is added to the flushingvessel. The piston pump is set to reverse and the flow rate controlleris set to 50%. The pump is then turned on and at least 500 cc of the 3%hydrogen peroxide solution is drawn to waste. Thereafter, the open endof the second piece of vacuum tubing is removed from the graduated flaskand placed it into the sterile flushing vessel. The drive is maintainedin the reverse position at 50%. The hydrogen peroxide is then allowed torecirculate for a minimum of 15 minutes.

The hydrogen peroxide is then decanted and approximately 3980 cc ofsterile water is added along with the entire contents of reconstitutedvials of Bacitracin and Polymyxin B to the flushing vessel. The flushingvessel is clearly labeled “antibiotic.” The piston pump is then set toreverse and the flow rate controller is set at 50%. The pump is turnedon and at least 500 cc of antibiotic solution is drawn to waste. Theopen end of the second piece of vacuum tubing is removed from thegraduated flask and placed into the sterile flushing vessel. The driveis maintained in the reverse position at 50%. The antibiotic solution isallowed to recirculate for a minimum of 15 minutes.

B. Plasticization:

The antibiotic solution is then decanted and approximately 4 liters of70% isopropyl alcohol/30% glycerin USP is added to the flushing vessel.The flushing vessel is clearly labeled as 70% IPA/30% glycerin USP. Thepiston pump is set to reverse and the flow rate controller is set to50%. The pump is turned on and at least 500 cc of IPA/glycerin USPsolution is drawn to waste.

The open end of the second piece of vacuum tubing is removed from thegraduated flask and placed into the sterile flushing vessel. The driveis maintained in the reverse position and the flow controller is set to50%. The IPA/glycerin USP is allowed to recirculate for a minimum of 30minutes. The IPA/glycerin USP solution is decanted and 4 liters of 30%glycerin USP in sterile water is added to the flushing vessel. Theflushing vessel is labeled as glycerin USP washing solution. The pistonpump is set to reverse and the flow rate controller is set to 50%. Thepump is turned on and at least 500 cc of washing solution is drawn towaste.

The open end of the second piece of vacuum tubing is removed from thegraduated flask and placed into the sterile flushing vessel. The driveis maintained in the reverse position and the flow rate controller isset to 50%. The washing solution is allowed to recirculate for a minimumof 15 minutes. Thereafter, the bone graft is removed from the flushingvessel and processed for freeze-drying as per standard operatingprocedure.

EXAMPLE 2 Processing of a Frozen Distal Femur

A. Cleaning and Processing:

A frozen distal femur is selected and all of the soft tissue andperiosteum is removed using sharp dissection techniques and periostealelevators. The graft is then transected to the desired length using aStryker® saw or band saw. Each bisected piece is not more than 30 cm inlength and is straight and contains no bone fragments. The surfacecartilage is then removed from the femoral condyle with either a scalpelblade, periosteal elevator, or osteotome. The processing instructionsdictate leaving the cartilage “on” when appropriate. Using a ⅜″ drillbit, the cut end of the shaft is drilled approximately 5 cm. Theinterior of the intramedullary canal is then throughly washed with thelavage system.

An intercalary fitting is then inserted by screwing the threaded,tapered end into the cut end of the graft. The vacuum tubing isassembled by securing one end of the tubing to the nipple end of theintercalary fitting. The other end of the tubing is secured to thepiston driven pump. Finally, another section of vacuum tubing is securedto the other side of the piston pump. Approximately 4000 cc of a 1:100dilution of the “Allowash™ Solution” is poured into the sterile flushingvessel. The “Allowash™ Solution” is prepared by adding 4 cc of cleaningreagent The flushing vessel is labeled as “Allowash™ Solution.” The openend of the second piece of vacuum tubing is placed into graduated flask.The piston pump is set to “reverse” and the flow rate controller is setto 50%. The pump is turned on and at least 500 cc of the first solvent(Allowash Solution) is drawn to waste. Thereafter, the open end of thesecond piece of vacuum tubing is removed from the graduated flask andplaced into the sterile flushing vessel. The drive is maintained in the“reverse” position at 50%. The Allowash Solution recirculates for aminimum of 15 minutes.

B. Plasticization:

The 1:100 dilution of the Allowash™ Solution is decanted andapproximately 4 liters of 3% hydrogen peroxide/30% glycerin USP is addedto the flushing vessel. The piston pump is set to reverse and the flowrate controller is set to 50%. The pump is turned on and at least 500 ccof the 3% hydrogen peroxide/glycerin USP solution is drawn to waste. Theopen end of the second piece of vacuum tubing is removed from thegraduated flask and placed into the sterile flushing vessel. The driveis maintained in the reverse position. The hydrogen peroxide/glycerinUSP is allowed to recirculate for a minimum of 15 minutes.

The hydrogen peroxide/glycerin USP is then decanted and approximately3980 cc of sterile water is added along with the entire contents ofreconstituted vials of Bacitracin and Polymyxin B prepared in a watersolution of 30% glycerin USP, to the flushing vessel. The flushingvessel is clearly labeled “antibiotic.” The piston pump is then set toreverse and the flow rate controller is set at 50%. The pump is turnedon and at least 500 cc of antibiotic solution is drawn to waste. Theopen end of the second piece of vacuum tubing is removed from thegraduated flask and placed into the sterile flushing vessel. The driveis maintained in the reverse position at 50%. The antibiotic solution isallowed to recirculate for a minimum of 15 minutes.

The antibiotic solution is then decanted and approximately 4 liters of70% isopropyl alcohol/30% glycerin USP is added to the flushing vessel.The flushing vessel is clearly labeled as 70% IPA/30% glycerin USP. Thepiston pump is set to reverse and the flow rate controller is set to50%. The pump is turned on and at least 500 cc of IPA/glycerin USPsolution is drawn to waste.

The open end of the second piece of vacuum tubing is removed from thegraduated flask and placed into the sterile flushing vessel. The driveis maintained in the reverse position and the flow controller is set to50%. The IPA/glycerin USP is allowed to recirculate for a minimum of 30minutes. The IPA/glycerin USP solution is decanted and 4 liters of 30%glycerin USP in sterile water is added to the flushing vessel. Theflushing vessel is labeled as glycerin USP washing solution. The pistonpump is set to reverse and the flow rate controller is set to 50%. Thepump is turned on and at least 500 cc of washing solution is drawn towaste.

The open end of the second piece of vacuum tubing is removed from thegraduated flask and placed into the sterile flushing vessel. The driveis maintained in the reverse position and the flow rate controller isset to 50%. The washing solution is allowed to recirculate for a minimumof 15 minutes. Thereafter, the bone graft is removed from the flushingvessel and processed for freeze-drying as per standard operatingprocedure.

EXAMPLE 3 Processing of a Frozen Distal Femur

A. Cleaning and Processing:

A frozen distal femur is selected and all of the soft tissue andperiosteum is removed using sharp dissection techniques and periostealelevators. The graft is then transected to the desired length using aStryker® saw or band saw. Each bisected piece is not more than 30 cm inlength and is straight and contains no bone fragments. The surfacecartilage is then removed from the femoral condyle with either a scalpelblade, periosteal elevator, or osteotome. The processing instructionsdictate leaving the cartilage “on” when appropriate. Using a ⅜″ drillbit, the cut end of the shaft is drilled approximately 5 cm. Theinterior of the intramedullary canal is then throughly washed with thelavage system.

An intercalary fitting is then inserted by screwing the threaded,tapered end into the cut end of the graft. The vacuum tubing isassembled by securing one end of the tubing to the nipple end of theintercalary fitting. The other end of the tubing is secured to thepiston driven pump. Finally, another section of vacuum tubing is securedto the other side of the piston pump. Approximately 4000 cc of a 1:100dilution of the “Allowash™ Solution” is poured into the sterile flushingvessel. The “Allowash™ Solution” is prepared by adding 4 cc of cleaningreagent. The flushing vessel is labeled as “Allowash™ Solution.” Theopen end of the second piece of vacuum tubing is placed into a graduatedflask. The piston pump is set to “reverse” and the flow rate controlleris set to 50%. The pump is turned on and at least 500 cc of the firstsolvent (Allowash Solution) is drawn to waste. Thereafter, the open endof the second piece of vacuum tubing is removed from the graduated flaskand placed into the sterile flushing vessel. The drive is maintained inthe “reverse” position at 50%. The Allowash Solution recirculates for aminimum of 15 minutes.

The 1:100 dilution of the Allowash™ Solution is then decanted andapproximately 4 liters of 3% hydrogen peroxide is added to the flushingvessel. The piston pump is set to reverse and the flow rate controlleris set to 50%. The pump is then turned on and at least 500 cc of the 3%hydrogen peroxide solution is drawn to waste. Thereafter, the open endof the second piece of vacuum tubing is removed from the graduated flaskand placed it into the sterile flushing vessel. The drive is maintainedin the reverse position at 50%. The hydrogen peroxide is then allowed torecirculate for a minimum of 15 minutes.

The hydrogen peroxide is then decanted and approximately 3980 cc ofsterile water is added along with the entire contents of reconstitutedvials of Bacitracin and Polymyxin B to the flushing vessel. The flushingvessel is clearly labeled “antibiotic.” The piston pump is then set toreverse and the flow rate controller is set at 50%. The pump is turnedon and at least 500 cc of antibiotic solution is drawn to waste. Theopen end of the second piece of vacuum tubing is removed from thegraduated flask and placed into the sterile flushing vessel. The driveis maintained in the reverse position at 50%. The antibiotic solution isallowed to recirculate for a minimum of 15 minutes.

The antibiotic solution is then decanted and approximately 4 liters of70% isopropyl alcohol (IPA) is added to the flushing vessel. Theflushing vessel is labeled as 70% IPA. The piston pump is set to reverseand the flow rate controller is set to 50%. The pump is turned on and atleast 500 cc of IPA solution is drawn to waste. The open end of thesecond piece of vacuum tubing is removed from the graduated flask andplaced into the sterile flushing vessel. The drive is maintained in thereverse position and the flow controller is set to 50%. The IPArecirculates for a minimum of 15 minutes. The IPA solution is thendecanted and 4 liters of sterile water is added to the flushing vessel.The flushing vessel is labeled as “washing solution.” The piston pump isset to reverse and the flow rate controller is set to 50%. The pump isturned on and at least 500 cc of washing solution is drawn to waste.

The open end of the second piece of vacuum tubing is removed from thegraduated flask and placed into the sterile flushing vessel. The driveis maintained in the reverse position and the flow rate controller isset to 50%. The washing solution recirculates for a minimum of 15minutes. The bone graft is removed from the flushing vessel andprocessed for freeze-drying as per standard operating procedure.

B. Plasticization:

The freeze-dried bone graft(s) are then placed into sterile glycerin USPsuch that they are totally immersed in the viscous glycerol. Vacuum (10to 500 mTorr, preferably 100 to 200 mTorr) is applied to the containeruntil bubbles cease to exit the bone graft (about 5 to 60 minutesdepending on the size and configuration of the bone graft, preferablyabout 20 to 30 minutes). The bone graft(s) are then removed from theglycerin USP solution and placed into an appropriate centrifugecontainer on top of a graft support.

The bone graft(s) are centrifuged at about 1000 to 2000 rpm until theglycerol ceases to exit the bone graft and accumulate in the bottom ofthe centrifuge container (usually 5 to 60 minutes depending on the sizeand configuration of the bone graft, preferably about 5 to 15 minutes).The bone graft(s) are then removed from their respective centrifugecontainers and packaged for distribution.

EXAMPLE 4 Processing Cloward Dowels

A. Cleaning and Processing:

Graft material is selected and all of the soft tissue and periosteum isremoved from the distal femur, proximal and distal tibia, and cartilageis removed from the site. The femur is transected 10-15 cm above thefemoral condyles and the distal femoral condyles are bisected. Transectthe proximal tibia 10-15 cm below the tibial plateau. The distal femuror proximal tibia is placed in a Pan-A-Vise™. This is accomplished byremoving a section of the diaphysis, allowing the vise jaws to grip thetissue securely. The Cloward set (12, 14,16, 18, or 20 mm) is thenassembled: 1. Place the extractor assembly within the cutter shaft, 2.Screw the cutter assembly onto the shaft with the aid of the Cloward setwrench, 3. Screw the set-point onto the extractor assembly, 4. Insertthe shaft of the Cloward set into the ⅜″ variable speed drill andtighten the chuck with the key. The set-point is then placed and lockedat the forward aspect of the cutter.

The apparatus is then placed on the tissue to be fashioned. Drilling iscommenced at a moderate speed. After the set-point has made a deep cutin the tissue, and the teeth have begun to cut into the tissue, drillingis stopped, and the set-point apparatus is unlocked. Drilling iscontinued using the marks created as a guide.

The Cloward(s) are then removed from the tissue block. A Stryker® saw orband saw is then used to remove the cut grafts after all have been cut.Any cartilage is then trimmed from the cortical face of the Cloward(s)using a scalpel and a # 10 blade. The distal end of the graft is thentrimmed perpendicular to the body of the graft with a band saw makingsure the fashioned graft is at least 15 mm long. The Cloward(s) arecleansed using pulsatile water apparatus. If the surface marrow is noteasily removed, dry spin the graft(s) at 2600 rpm for 3 minutes.

The Cloward(s) are then placed in a sterile container with hydrogenperoxide (3%) at 37 to 44° C., The container is sealed and the containeris placed into the centrifuge. The centrifuge is then balanced. Thegrafts are then centrifuged at 2600 rpm for 15 minutes. The tissue isremoved from the centrifuge and the grafts are placed into an ultrasoniccleaner. Equal volumes of Allowash™ Solution, hydrogen peroxide (3%),and antibiotics are added to the ultrasonic cleaner and sonicate thetissue at 37-44° C. for a minimum of 1 hour. Thereafter, the tissue isremoved from the ultrasonic cleaner.

The mixture is decanted and a sterile glass container is filled withfresh 3% hydrogen peroxide. The grafts are then placed in the container,the top is sealed and the container is taken to the large ultrasoniccleaner. The grafts are then sonicated for 90 minutes. Thereafter, thegrafts are incubated overnight at 37-44° C. (minimum of 6 hours,preferably 12 to 18 hours).

B. Plasticization:

After incubation, the hydrogen peroxide is decanted and the basin isfilled with 70% isopropyl alcohol/30% glycerin USP and the grafts areincubated at room temperature for a minimum of 30 minutes. Thereafter,the isopropyl alcohol/glycerin USP solution is decanted and thecontainer is filled with warm 30% glycerin USP in water. The grafts areincubated for a minimum of 30 minutes. Methods of incubation include forexample: soaking.

The glycerin solution is then decanted and the Cloward dowels areremoved from the container. The Cloward dowels are then placed into asterile container. The container is sealed and placed into thecentrifuge. The centrifuge is balanced and the grafts are centrifugedfor 3-5 minutes to dry, and the remaining solution is removed.

The width and length of the Cloward(s) are measured, graftidentification numbers are assigned, and the information is recorded onthe “Tissue Processing Log Worksheet”. One graft is then placed into aglass, 120 cc bottle and the printed label is affixed with the uniquenumeric designator. This step is repeated until all deposits arebottled. The bottled grafts are either frozen and packaged, or frozenand freeze-dried and packaged.

EXAMPLE 5 Processing Cloward Dowels

A. Cleaning and Processing:

Graft material is selected and all of the soft tissue and periosteum isremoved from the distal femur, proximal and distal tibia, and cartilageis removed from the site. The femur is transected 10-15 cm above thefemoral condyles and the distal femoral condyles are bisected. Transectthe proximal tibia 10-15 cm below the tibial plateau. The distal femuror proximal tibia is placed in a Pan-A-Vise™. This is accomplished byremoving a section of the diaphysis, allowing the vise jaws to grip thetissue securely. The Cloward set (12, 14, 16, 18, or 20 mm) is thenassembled: 1. Place the extractor assembly within the cutter shaft, 2.Screw the cutter assembly onto the shaft with the aid of the Cloward setwrench, 3. Screw the set-point onto the extractor assembly, 4. Insertthe shaft of the Cloward set into the ⅜″ variable speed drill andtighten the chuck with the key. The set-point is then placed and lockedat the forward aspect of the cutter.

The apparatus is then placed on the tissue to be fashioned. Drilling iscommenced at a moderate speed. After the set-point has made a deep cutin the tissue, and the teeth have begun to cut into the tissue, drillingis stopped, and the set-point apparatus is unlocked. Drilling iscontinued using the marks created as a guide.

The Cloward(s) are then removed from the tissue block. A Stryker® saw orband saw is then used to remove the cut grafts after all have been cut.Any cartilage is then trimmed from the cortical face of the Cloward(s)using a scalpel and a # 10 blade. The distal end of the graft is thentrimmed perpendicular to the body of the graft with a band saw makingsure the fashioned graft is at least 15 mm long. The Cloward(s) arecleansed using pulsatile water apparatus. If the surface marrow is noteasily removed, dry spin the graft(s) at 2600 rpm for 3 minutes.

B. Plasticization:

The Cloward(s) are then placed in a sterile container with hydrogenperoxide (3%) and glycerin USP (30%) at 37 to 44° C. The container issealed and the container is placed into the Centrifuge. The centrifugeis then balanced. The grafts are then centrifuged at 2600 rpm for 15minutes. The tissue is removed from the centrifuge and the grafts areplaced into an ultrasonic cleaner. Equal volumes of Allowash™ Solution,hydrogen peroxide (3%), 30% glycerin USP, and antibiotics are added tothe ultrasonic cleaner and the tissue is sonicated at 37-44° C. for aminimum of 1 hour. Thereafter, the tissue is removed from the ultrasoniccleaner.

The mixture is decanted and a sterile glass container is filled withfresh 3% hydrogen peroxide/30% glycerin USP. The grafts are then placedin the container, the top is sealed and the container is taken to thelarge ultrasonic cleaner. The grafts are then sonicated for 90 minutes.Thereafter, the grafts are incubated overnight at 37-44° C. (minimum of6 hours, preferably 12 to 18 hours).

After incubation, the hydrogen peroxide is decanted and the basin isfilled with 70% isopropyl alcohol/30% glycerin USP and the grafts areincubated at room temperature for a minimum of 30 minutes. Thereafter,the isopropyl alcohol/glycerin USP solution is decanted and thecontainer is filled with warm 30% glycerin USP in water. The grafts areincubated for a minimum of 30 minutes. Methods of incubation include forexample: soaking and mild agitation.

The glycerin solution is then decanted and the Cloward dowels areremoved from the container. The Cloward dowels are then placed into asterile container. The container is sealed and placed into thecentrifuge. The centrifuge is balanced and the grafts are centrifugedfor 3-5 minutes to dry, and the remaining solution is removed.

The width and length of the Cloward(s) are measured, graftidentification numbers are assigned, and the information is recorded onthe “Tissue Processing Log Worksheet”. One graft is then placed into aglass, 120 cc bottle and the printed label is affixed with the uniquenumeric designator. This step is repeated until all deposits arebottled. The bottled grafts are either frozen and packaged, or frozenand freeze-dried and packaged.

EXAMPLE 6 Processing Cloward Dowels

A. Cleaning and Processing:

Graft material is selected and all of the soft tissue and periosteum isremoved from the distal femur, proximal and distal tibia, and cartilageis removed from the site. The femur is transected 10-15 cm above thefemoral condyles and the distal femoral condyles are bisected. Transectthe proximal tibia 10-15 cm below the tibial plateau. The distal femuror proximal tibia is placed in a Pan-A-Vise™. This is accomplished byremoving a section of the diaphysis, allowing the vise jaws to grip thetissue securely. The Cloward set (12, 14, 16, 18, or 20 mm) is thenassembled: 1. Place the extractor assembly within the cutter shaft, 2.Screw the cutter assembly onto the shaft with the aid of the Cloward setwrench, 3. Screw the set-point onto the extractor assembly, 4. Insertthe shaft of the Cloward set into the ⅜″ variable speed drill andtighten the chuck with the key. The set-point is then placed and lockedat the forward aspect of the cutter.

The apparatus is then placed on the tissue to be fashioned. Drilling iscommenced at a moderate speed. After the set-point has made a deep cutin the tissue, and the teeth have begun to cut into the tissue, drillingis stopped, and the set-point apparatus is unlocked. Drilling iscontinued using the marks created as a guide.

The Cloward(s) are then removed from the tissue block. A Stryker® saw orband saw is then used to remove the cut grafts after all have been cut.Any cartilage is then trimmed from the cortical face of the Cloward(s)using a scalpel and a # 10 blade. The distal end of the graft is thentrimmed perpendicular to the body of the graft with a band saw makingsure the fashioned graft is at least 15 mm long. The Cloward(s) arecleansed using pulsatile water apparatus. If the surface marrow is noteasily removed, dry spin the graft(s) at 2600 rpm for 3 minutes.

The Cloward(s) are then placed in a sterile container with hydrogenperoxide (3%) at 37 to 44° C. The container is sealed and the containeris placed into the centrifuge. The centrifuge is then balanced. Thegrafts are then centrifuged at 2600 rpm for 15 minutes. The tissue isremoved from the centrifuge and the grafts are placed into an ultrasoniccleaner. Equal volumes of Allowash™ Solution, hydrogen peroxide (3%),and antibiotics are added to the ultrasonic cleaner and sonicate thetissue at 37-44° C. for a minimum of 1 hour. Thereafter, the tissue isremoved from the ultrasonic cleaner.

The mixture is decanted and a sterile glass container is filled withfresh 3% hydrogen peroxide. The grafts are then placed in the container,the top is sealed and the container is taken to the large ultrasoniccleaner. The grafts are then sonicated for 90 minutes. Thereafter, thegrafts are incubated overnight at 37-44° C. (minimum of 6 hours,preferably 12 to 18 hours).

After incubation, the hydrogen peroxide is decanted and the basin isfilled with 70% isopropyl alcohol and the grafts are incubated at roomtemperature for a minimum of 30 minutes. Thereafter, the isopropylalcohol is decanted and the container is filled with warm sterile water.The grafts are incubated for a minimum of 30 minutes. Methods ofincubation include for example: soaking and mild agitation.

The wash solution is then decanted and the Cloward dowels are removedfrom the container. The Cloward dowels are then placed into a sterilecontainer. The container is sealed and placed into the centrifuge. Thecentrifuge is balanced. The grafts are then centrifuged for 3-5 minutesto dry and the remaining solution is removed.

The width and length of the Cloward(s) are measured, graftidentification numbers are assigned, and the information is recorded onthe “Tissue Processing Log Worksheet”. One graft is then placed into aglass, 120 cc bottle and the printed label is affixed with the uniquenumeric designator. This step is repeated until all deposits arebottled. The bottled grafts are either frozen and packaged, or frozenand freeze-dried and packaged.

B. Plasticization:

Viscous glycerol is then added to each bottle sufficient to cover thegraft and vacuum (10 to 500 mTorr) is applied to each bottle until theair ceases to exit the grafts (usually 5-20 minutes depending on grafttype). The grafts are then removed from the bottles and placed into acentrifuge container. The grafts are centrifuged for 15-30 minutes oruntil glycerol ceases to exit the grafts and accumulate in the spacebelow the grafts. The bottled grafts are either packaged or placed undervacuum and packaged.

EXAMPLE 7 Processing Cloward Dowels

A. Cleaning and Processing:

Graft material is selected and all of the soft tissue and periosteum isremoved from the distal femur, proximal and distal tibia, and cartilageis removed from the site. The femur is transected 10-15 cm above thefemoral condyles and the distal femoral condyles are bisected. Transectthe proximal tibia 10-15 cm below the tibial plateau. The distal femuror proximal tibia is placed in a Pan-A-Vise™. This is accomplished byremoving a section of the diaphysis, allowing the vise jaws to grip thetissue securely. The Cloward set (12, 14, 16, 18, or 20 mm) is thenassembled: 1. Place the extractor assembly within the cutter shaft, 2.Screw the cutter assembly onto the shaft with the aid of the Cloward setwrench, 3. Screw the set-point onto the extractor assembly, 4. Insertthe shaft of the Cloward set into the ⅜″ variable speed drill andtighten the chuck with the key. The set-point is then placed and lockedat the forward aspect of the cutter.

The apparatus is then placed on the tissue to be fashioned. Drilling iscommenced at a moderate speed. After the set-point has made a deep cutin the tissue, and the teeth have begun to cut into the tissue, drillingis stopped, and the set-point apparatus is unlocked. Drilling iscontinued using the marks created as a guide.

The Cloward(s) are then removed from the tissue block. A Stryker® saw orband saw is then used to remove the cut grafts after all have been cut.Any cartilage is then trimmed from the cortical face of the Cloward(s)using a scalpel and a # 10 blade. The distal end of the graft is thentrimmed perpendicular to the body of the graft with a band saw makingsure the fashioned graft is at least 15 mm long. The Cloward(s) arecleansed using pulsatile water apparatus. If the surface marrow is noteasily removed, dry spin the graft(s) at 2600 rpm for 3 minutes.

B. Plasticization:

The Cloward(s) are then placed in a sterile container with hydrogenperoxide (3%)/glycerin USP 30% at 37 to 44° C. The container is sealedand the container is placed into the centrifuge. The centrifuge is thenbalanced. The grafts are then centrifuged at 2600 rpm for 15 minutes.The tissue is removed from the centrifuge and the grafts are placed intoan ultrasonic cleaner. Equal volumes of Allowash™ Solution, hydrogenperoxide (3%), and antibiotics are added to the ultrasonic cleaner andsonicate the tissue at 37-44° C. for a minimum of 1 hour. Thereafter,the tissue is removed from the ultrasonic cleaner.

The mixture is decanted and a sterile glass container is filled withfresh 3% hydrogen peroxide. The grafts are then placed in the container,the top is sealed and the container is taken to the large ultrasoniccleaner. The grafts are then sonicated for 90 minutes. Thereafter, thegrafts are incubated overnight at 37-44° C. (minimum of 6 hours,preferably 12 to 18 hours).

After incubation, the hydrogen peroxide is decanted and the basin isfilled with 70% isopropyl alcohol/30% glycerin USP and the grafts areincubated at room temperature for a minimum of 30 minutes. Thereafter,the isopropyl alcohol/glycerin USP solution is decanted and thecontainer is filled with warm 30% glycerin USP in sterile water. Thegrafts are incubated for a minimum of 30 minutes. Methods of incubationinclude for example: soaking and mild agitation.

The solution is then decanted and the Cloward dowels are removed fromthe container. The Cloward dowels are then placed into a sterilecontainer. The container is sealed and placed into the centrifuge. Thecentrifuge is balanced. The grafts are then centrifuged for 3-5 minutesto dry and the remaining solution is removed.

The width and length of the Cloward(s) are measured, graftidentification numbers are assigned, and the information is recorded onthe “Tissue Processing Log Worksheet”. One graft is then placed into aglass, 120 cc bottle and the printed label is affixed with the uniquenumeric designator. This step is repeated until all deposits arebottled. The bottled grafts are either frozen and packaged, or frozenand freeze-dried and packaged.

EXAMPLE 8 Processing of an Iliac Crest Wedge

A. Cleaning and Processing:

The soft tissue, periosteum, and cartilage is removed from an ilium. Theilium is placed in a Pan-A-Vise™ by removing a section of the ilium,allowing the vise jaws to grip the tissue securely. A Stryker saw isassembled with parallel cutting blades (12, 14, 16, 18, or 20 mm). Theset-point at the forward aspect of the cutter is placed and locked. Theapparatus is placed on the tissue to be fashioned and cutting is begunat a moderate speed.

After the set-point has made a deep cut in the tissue, and the teethhave begun to cut into the tissue, cutting is stopped, and the set-pointapparatus is checked. Cutting is continued using the marks created as aguide. A Stryker® saw or band saw is then used to remove the cut graftsafter all have been cut.

Any cartilage is trimmed from the cortical face of the grafts(s) using ascalpel and a # 10 blade. The distal end of the graft perpendicular tothe body of the graft is trimmed with a band saw making sure thefashioned graft is at least 15 mm long. The Grafts is then cleansedusing a pulsatile water apparatus. If the surface marrow is not easilyremoved, the graft(s) is dry spun at 2600 rpm for 3 minutes.

B. Plasticization:

The Iliac Crest Wedge(s) are then placed in a sterile container withhydrogen peroxide (3%) and glycerin USP (30%) at 37 to 44° C. Thecontainer is sealed and placed into the centrifuge. The centrifuge isbalanced. The grafts are then centrifuged at 2600 rpm for 15 minutes.The tissue is removed from the centrifuge and the grafts are placed intothe ultrasonic cleaner. Equal volumes of Allowash™ Solution, hydrogenperoxide (3%), glycerin USP (30%), and antibiotics are added to theultrasonic cleaner, and the grafts are sonicated at 37-44° C. for aminimum of 1 hour.

The tissue is then removed from the ultrasonic cleaner. The mixture isdecanted and a sterile glass container is filled with fresh 3% hydrogenperoxide/30% glycerin USP. The grafts are placed in the container, thetop is sealed and the container is taken to a large ultrasonic cleaner.The grafts are sonicated for 90 minutes. Thereafter, the grafts areincubated overnight at 37-44° C. (minimum of 6 hours, preferably for 12to 18 hours). Methods of incubation include for example: soaking andmild agitation.

The hydrogen peroxide/glycerin USP is then decanted and the basin isfilled with 70% isopropyl alcohol/30% glycerin USP. The grafts are thenincubated at room temperature for a minimum of 30 minutes. The isopropylalcohol/glycerin USP solution is then decanted and the container isfilled with warm 30% glycerin USP in water. The grafts are incubated fora minimum of 30 minutes.

The glycerin USP solution is then decanted and the Iliac Crest Wedgesare removed from the container. The Iliac Crest Wedges are then placedinto a sterile container. The container is sealed and placed into thecentrifuge. The centrifuge is balanced and the grafts are centrifugedfor 3-5 minutes to dry and the remaining solution is removed.

The width and length of the Wedges are measured, graft identificationnumbers are assigned, and the information is recorded on the “TissueProcessing Log Worksheet”. One graft is then placed into a glass, 120 ccbottle and the printed label is affixed with the unique numericdesignator. This step is repeated until all deposits are bottled. Thebottled grafts are either frozen and packaged, or frozen andfreeze-dried and packaged.

EXAMPLE 9 Processing of Fascia Lata

A. Cleaning and Processing:

Any remaining muscle tissue is removed from the fascia lata. The fasciais placed with the subcutaneous layer uppermost, on a clean, drapetowel. Using blunt dissection techniques, all of the fat and extraneoussoft tissue is removed from the graft material. The graft is kept moistwith sterile water to prevent desiccation during processing.

Any torn fibers are removed from the edges of the graft material, and agraft rectangular in shape is created. The graft(s) are then placed in abasin containing a 1:100 dilution of Allowash™ Solution or othersurfactant(s) for at least 15 minutes. The basin is labeled as Allowash™Solution. The time of exposure is recorded on the Tissue Processing LogWorksheet.

B. Plasticization:

The graft(s) are placed into an empty basin labeled “Rinse”. Thegraft(s) are rinsed three time with copious amounts of sterile water toremove any residual detergents. Any sterile water which accumulates inthe Rinse basin is discarded. The number of rinses is recorded on theTissue Processing Log Worksheet. The fashioned graft(s) are then placedin the basin containing U.S.P. grade 70% isopropyl alcohol containing30% glycerin USP for 2-5 minutes. The basin is labeled IPA/Glycerin. Thetime of exposure to the alcohol/glycerin USP solution is recorded in theTissue Processing Log Worksheet.

The graft(s) are then placed into the basin containing the antibioticsolution in 30% glycerin USP for at least 15 minutes. The basin islabeled as Antibiotics/Glycerin USP. The exposure time to theantibiotics/glycerin USP is recorded on the Tissue Processing LogWorksheet. The graft(s) are then thoroughly soaked by immersing eachdeposit into sterile 30% glycerin USP in deionized/distilled water for aminimum of 5 minutes to remove excess antibiotics. Enough sterileglycerin USP solution is needed to cover the graft(s). The basin islabeled as Rinse. The time of exposure to the glycerin USP rinsesolution is recorded on the Tissue Processing Log Worksheet.

The fashioned graft(s) are then placed on sterile fine mesh gauze, andthe gauze is trimmed to just beyond the edges of the graft. The widthand length of the graft(s) is measured to the nearest tenth of acentimeter. The graft(s) are assigned identification numbers and thisinformation is recorded on the Tissue Processing Log Worksheet. Thegraft and gauze is then rolled into a tube and graft material is thenplaced into glass, 120 ml bottles, and the printed label is affixed withthe unique numeric designator. This step is repeated until all depositsare bottled. The graft material is now ready for wrapping andfreeze-drying or dehydrating.

EXAMPLE 10 Processing Pericardium

A. Cleaning and Processing:

The pericardial tissue is rinsed of any blood or pericardial fluid insterile water in the basin labeled Rinse. The pericardium is then placedon a clean drape towel. Using blunt dissection techniques, all of thefat and extraneous soft tissue is removed from the graft material. Thegraft is kept moist with sterile water to prevent desiccation duringprocessing. Any torn fibers are removed from the edges of the graftmaterial, and a graft rectangular in shape is created. The graft(s) arethen placed in a basin containing a 1:100 dilution of Allowash Solutionor other surfactant(s) for at least 15 minutes. The basin is labeled asAllowash™ Solution. The time of exposure is recorded on the TissueProcessing Log Worksheet.

The graft(s) are placed into an empty basin labeled “Rinse”. Thegraft(s) are rinsed three time with copious amounts of sterile water toremove any residual detergents. Any sterile water which accumulates inthe Rinse basin is discarded. The number of rinses is recorded on theTissue Processing Log Worksheet. The fashioned graft(s) are then placedin the basin containing U.S.P. grade 70% isopropyl alcohol containing30% glycerin USP for 2-5 minutes. The basin is labeled IPA/Glycerin. Thetime of exposure to the alcohol/glycerin USP solution is recorded in theTissue Processing Log Worksheet.

The graft(s) are then placed into the basin containing the antibioticsolution in 30% glycerin USP for at least 15 minutes. The basin islabeled as Antibiotics/Glycerin USP. The exposure time to theantibiotics/glycerin USP is recorded on the Tissue Processing LogWorksheet. The graft(s) are then thoroughly soaked by immersing eachdeposit into sterile 30% glycerin USP in deionized/distilled water for aminimum of 5 minutes, preferably from 10 to 15 minutes, to remove excessantibiotics. Enough sterile glycerin USP solution is needed to cover thegraft(s). The basin is labeled as Rinse. The time of exposure to theglycerin USP rinse solution is recorded on the Tissue Processing LogWorksheet.

The fashioned graft(s) are then placed on sterile fine mesh gauze, andthe gauze is trimmed to just beyond the edges of the graft. The widthand length of the graft(s) is measured to the nearest tenth of acentimeter. The graft(s) are assigned identification numbers and thisinformation is recorded on the Tissue Processing Log Worksheet. Thegraft and gauze is then rolled into a tube and graft material is thenplaced into glass, 120 ml bottles, and the printed label is affixed withthe unique numeric designator. This step is repeated until all depositsare bottled. The graft material is now wrapped and placed in a freezedryer or dehydrated.

All of the publications and patent applications cited herein are herebyincorporated by reference into the present disclosure. It will beappreciated by those skilled in the art that various modifications canbe made without departing from the essential nature thereof It isintended to encompass all such modifications within the scope of theappended claims.

What is claimed:
 1. A plasticized, load-bearing, bone graft, comprising:a cleaned, non-demineralized, load-bearing, bone graft impregnated withone or more plasticizers, said load-bearing bone graft is notfreeze-dried.
 2. A plasticized, load-bearing, bone graft, comprising: acleaned, non-demineralized, load-bearing, bone graft and one or moreplasticizers, said load-bearing bone graft is not freeze-dried.
 3. Theplasticized bone graft of any one of claims 1 or 2, said bone graft issuitable for direct transplantation into a human without rehydration ofsaid bone graft.
 4. The plasticized bone graft of any one of claims 1 or2, said bone graft comprising a member selected from the groupconsisting of an essentially intact bone, a substantial portion of awhole bone, and a cut bone.
 5. The plasticized bone graft of any one ofclaims 1 or 2, said bone graft comprises allogenic or xenogenic bone. 6.A method for producing a plasticized, load-bearing, bone graft,comprising: impregnating a cleaned, non-demineralized, load-bearing,bone graft with one or more plasticizers to produce a plasticized,load-bearing, bone graft, said load-bearing bone graft is notfreeze-dried.
 7. The method of claim 6, said cleaned bone graft isessentially free from bone marrow elements.